1. Field of the Invention
This invention relates to peptides that increase the efficiency of gene delivery into cells and tissues and methods pertaining to the use of the peptides for delivering nucleic acids into cells or tissues. More specifically, this invention relates to amino acid sequences that mediate effective delivery of nucleic acids into cells and tissues and methods for delivering nucleic acids into cells or tissues using the peptides.
2. General Background and State of the Art
Recently, as the fields of gene therapy and molecular biology have developed rapidly, an urgent need has emerged to effectively deliver nucleic acids into cells or tissues. To be useful in gene therapy and antisense therapy as well as in vitro cell line experimentation, an effective amount of nucleic acids must be delivered into the target cells or tissue. At the same time, the nucleic acid delivery method should induce less immune response or cytotoxicity to the host. Also, availability of large-scale production of delivered materials, or vehicles, is important for the success of gene based therapy. Presently, gene delivery can be broadly divided into viral and non-viral delivery. For virus-mediated gene delivery, retrovirus, adenovirus, and adeno-associated virus and so on have been used for the transfer of nucleic acids into cells or tissues. Virus-mediated gene delivery has drawbacks including narrow range of cell infectivity, immune response to viruses, difficulty of large-scale production of viral vectors, etc. (Yibin Wang et al., DDT. 5(1), 2000; Joanne T. Douglas. et al., Science & medicine 44-52 (March/April), 1997).
For non-viral gene delivery, many different agents or methods such as liposomes, polymers, calcium phosphate, electroporation, and micro-injection have been used (Saghir Akhtar et al., Adv. Drug Deliv. Rev. 44:3-21; Irina Lebedeva et al., Eur. J. Pharm. Biopharm. 50:101-119, 2000; Ch. Garcia-Chaumont et al., Pharmacol. Ther. 76:151-161, 2000). Preparation of non-viral vectors is relatively easy such that large-scale production of vectors can be done to accommodate preclinical or clinical trials. Non-viral vectors are also known to induce less immune response to the vectors, allowing repeated administration of the vectors to the host. For these reasons, gene therapy using non-viral vectors has become popular (Colin W. Pouton et al., Adv. Drug Deliv. Rev. 46:187-20, 2001).
Among the non-viral vectors developed to date, liposomes are the most frequently used gene transfer vehicle and are available commercially. In order to take advantage of the negatively charged nature of cell surface, a large number of liposomes are cationic, or positively charged. Cationic liposomes, complexed with nucleic acids or DNA, electrostatically interact with the cell surface, and the complexes are then known to be endocytosed into the cell cytoplasm. Whereas cationic liposomes mediate gene delivery effectively into cells in vitro, gene delivery in an in vivo system is quite limited as compared to viral vectors. Furthermore, the efficiency of gene delivery using cationic liposomes is generally dependent on the size of nucleic acids, cell lines and so on, even in an in vitro system. In addition, increased amounts of nucleic acids are known to induce cytotoxicity to cells (Saghir Akhtar et al., Adv. Drug Deliv. Rev. 44:3-21, 2000: Irina Lebedeva et al., Eur. J. Pharm. Biopharm. 50:101-119, 2000).
Cationic polymers have been used to increase the efficiency of gene delivery into cells. By manipulating the number of positively charged amine group, they are able to bind strongly with nucleic acids, and also interact with the cell, so that the required amount of the polymers as compared to that of cationic liposomes can be reduced. However, cytotoxicity, and insolubility of cationic polymers in aqueous solutions should be resolved in order to be useful as an effective gene delivery vehicle (Dan Luo et al., Nat. biotech. 18:33-37; Saghir Akhtar et al., Adv. Drug Deliv. Rev. 44:3-21, 2000). Recently, a biodegradable polymer that dissolves well in water has been developed but no data is available regarding its safety in vivo (Sang-Oh Han et al., Bioconjugate Chem. 12:337-345, 2001; Otmane Boussif et al., Proc. Natl. Acd. Sci. 92:7297-7301, 1995).
The present invention was developed according to the above need for improvement of a nucleic acid delivery system. The present invention provides a gene delivery system that includes peptides that enhance nucleic acid uptake by cells. In addition, the present invention provides methods by which nucleic acids are delivered into cells or tissue using the above mentioned peptides.